Action of mung bean nuclease on supercoiled PM2 DNA.

Single strand specific mung bean nuclease was used to probe for regions of altered secondary structure in supercoiled PM2 DNA. Supercoiled DNA is cleaved greater than or equal to 10,000 times faster than the relaxed topoisomer. Catalytic quantities of enzyme convert supercoiled DNA to both nicked-circular and unit length linear forms at pH 5 but to predominantly the nicked-circular form near neutral pH. At the elevated enzyme concentrations required to cleave relaxed DNA, unit length linear DNA and smaller fragments are produced from pH 5 to 7. One nick per supercoiled DNA molecule is introduced at pH 6.6. The nicks are repairable by DNA ligase and are not strand-specific. Snake venom phosphodiesterase selectively cleaves the strand opposite the nicks, permitting restriction endonuclease mapping. The nicks occur at three specific sites. Sites at 0.75 and 0.76 map units are cleaved with equal frequency, while a site at 0.82 is cleaved less frequently. The former sites map near one of the eight known early denaturation regions of PM2 DNA, while the latter does not. Since most early denaturation sites are not cleaved, sites other than these dA + dT-rich regions may be the preferred locations of strand unwinding and separation in supercoiled PM2 DNA.

Single strand specific mung bean nuclease was used to probe for regions of altered secondary structure in supercoiled PM2 DNA. Supercoiled DNA is cleaved ~1 0 , 0 0 0 times faster than the relaxed topoisomer. Catalytic quantities of enzyme convert supercoiled DNA t o both nicked-circular and unit length linear forms at pH 5 but to predominantly the nicked-circular form near neutral pH. At the elevated enzyme concentrations required to cleave relaxed DNA, unit length linear DNA and smaller fragments are produced from pH 5 to 7. One nick per supercoiled DNA molecule is introduced at pH 6.6. The nicks are repairable by DNA ligase and are not strand-specific. Snake venom phosphodiesterase selectively cleaves the strand opposite the nicks, permitting restriction endonuclease mapping. The nicks occur at three specific sites. Sites at 0.75 and 0.76 map units are cleaved with equal frequency, while a site at 0.82 is cleaved less frequently. The former sites map near one of the eight known early denaturation regions of PM2 DNA, while the latter does not. Since most early denaturation sites are not cleaved, sites other than these dA + dT-rich regions may be the preferred locations of strand unwinding and separation in supercoiled PM2 DNA.
An endonuclease isolated from mung bean sprouts (1) preferentially cleaves denatured as opposed to native DNA (2) and thus has been called "single strand specific." Mung bean nuclease has been purified to homogeneity (3) and the activity on various regions of DNA characterized. Known singlestranded regions of DNA such as single-stranded tails (4) and internal single-stranded gaps (5) are preferentially cleaved, while nicks are relatively resistant. Transient, localized unwinding of duplex DNA structure may result in susceptible sites both within (2,5,6) and at the ends ( 7 ) of a linear molecule. Hydrolysis at these transiently single-stranded regions is slow relative to that of single-stranded DNA and is highly dependent on reaction conditions which affect the secondary structure of DNA (2,5,7 ) . Model DNA heteroduplexes whose mismatched bases may be accommodated in a stacked helical structure are cleaved at extremely low efficiency when only one base is mismatched and at greater efficiency as the number of adjacent mismatched bases is increased (8).
Closed circular duplex DNA can be isolated in a supercoiled form which is highly reactive to enzymes, proteins, and chemical agents which show a preference for single-stranded DNA (for reviews, see Refs. 9 and 10). The single strand character of this DNA is a consequence of torsional strain which, at sufficiently high negative superhelical density, promotes unwinding of helical twists (10, 11). In the absence of strand breakage, the unwinding of one turn of the double helix allows the untwisting of one negative supercoil (12). Negative supercoiling of DNA in prokaryotes is essential for cell growth and may be required to promote the strand unwinding and separation which occur during DNA replication, transcription, and recombination (for reviews, see Refs. 13 and 14). Thus, it is likely that some sites in negatively supercoiled DNA which are recognized by single strand specific agents are of biological importance.
We wanted to assess the feasibility of using mung bean nuclease to probe the structure of negatively supercoiled DNA near neutral pH, away from the acidic pH optimum of the enzyme. Neutral pH is advantageous since it more closely approximates the pH that both DNA and proteins which interact with DNA experience in uiuo. In addition, spontaneous nicking of DNA near neutral pH is negligible compared to that at acidic pH. At acidic pH, mung bean nuclease introduces a limited number of nonrandom cleavages in both supercoiled (15) and linear (5) forms of PM2 DNA. The locations of these sites were not determined.
In this paper, we demonstrate that mung bean nuclease is catalytically active on supercoiled PM2 DNA near neutral pH. We quantify the preference for the supercoiled over the relaxed topoisomer as a function of pH and characterize the products formed. We map the nicks introduced into supercoiled PM2 DNA near neutral pH utilizing venom phosphodiesterase as a reagent to selectively cleave the strand opposite the nicks. Finally, we compare the locations of single strand character detected by mung bean nuclease to the locations of the known early melting sites in PM2 DNA (16). solution a t 65 "C for 15 m i n . mM NaC1). Mung bean nuclease units refer to the standard assay procedure (3) using denatured salmon sperm DNA. The mung bean nuclease diluent contained 10 m~ Tris-HC1, pH 7.0,O.l m~ serine, 1 p~ zinc acetate, and 0.005% Triton X-100 (5). Two microliters of the appropriate enzyme dilution were added to the reaction mixture (18 p l ) containing 0.4 pg of PM2 DNA (2.0 pg where indicated) in a 1.5-ml conical polypropylene tube at 37 "C. Incubation time was 10 min unless otherwise indicated. For fluorometric analysis, the reaction was terminated by transferring 0.1-0.2 ml of ethidium bromide solution (see under "Fluorometric Assay" below) from a cuvette containing 1.0 ml of ethidium bromide solution to the reaction tube and then transferring the mixture back to the same cuvette. The reaction mixture was then rinsed once with the DNA/ethidium bromide mixture, and the mixture was returned to the cuvette. For gel electrophoretic analysis, reactions were terminated as indicated either below or in the figure legends. Linearization of Nicked-Circular DNA with Venom Phosphodiesterase-The mung bean nuclease reaction was terminated by placing the reaction mixture on ice. Conditions for phosphodiesterase digestion were established by addition of an equal volume of 40 mM Tris, 2 mM magnesium acetate, 0.01% Triton X-100, pH 11.7 (pH adjusted by addition of NaOH). The final pH is 9.2.One-ninth volume of phosphodiesterase (0.015 u n i t / d in diluent consisting of 25 mM Tris-HC1,0.5 m~ magnesium acetate, 0.00625% Triton X-100, pH 9.2) was added, and the mixture was incubated at 37 "C for 10 min. Control reactions without phosphodiesterase showed no further nicking of DNA by mung bean nuclease under these conditions. Phosphodiesterase was irreversibly inactivated by adding Yi volume 0.1 M Na2EDTA (pH 7). The reaction mixture was then placed on ice and samples taken for electrophoresis.
Phosphodiesterase Assay-Phosphodiesterase was assayed using bis-p-nitrophenyl phosphate (5 m~ bis-p-nitrophenyl phosphate, 10 mM MgCI,, 0.10 M Tris-HC1, pH 9.0, 1.0-ml volume) as substrate. After 5 min incubation a t 37 "C, the reaction was stopped with 2 ml of 0.1 M NaOH, 10 mM EDTA. The absorbance at 400 nm (A4w) is determined and corrected for the minus enzyme blank. One unit is defined as the quantity of enzyme which liberates 1 pmol of pnitrophenol per min and is equal to 0.034 X A 4~. Digestion of Linearized DNA with Restriction Endonucleases-After phosphodiesterase treatment, reaction conditions were adjusted for digestion with Hpa I1 (Msp I) by lowering the pH to 7.4 with 0.17 M acetic acid and addition of magnesium acetate to 17 m. Digestion of 2 pg of DNA in 60 pl was performed with 7.5 units (New England Biolabs) of enzyme at 37 "C for 2 h. A subsequent digestion with Pst I was performed by diluting 6 pJ of Hpa 11-restricted DNA (0.2 pg) with an equal volume of 36 m~ Tris, 30 mM potassium phosphate, 1 m~ NaZEDTA, incubating with 1 unit of Pst I for 30 min at 37 "C, and keeping on ice for 30 min.
For Hind111 digestions, pH and Mg2+ were adjusted as for Hpa I1 digestion but, in addition, the solution was made 50 m~ in NaCl. Digestion of 2 pg of DNA in 60 pl was performed at 37 "C for 3 h by adding 7.5 units of enzyme both at zero time and 1.5 h.
Fluorometric Assay for Endonuclease Activity on Covalently Closed Circular DNA-The assay monitors the conversion of covalently closed circular DNA (supercoiled or relaxed) to open forms (nicked-circular, linear). Covalently closed forms of DNA renature after heating in a pH 12 solution of ethidium bromide and cooling, while open forms do not (19). Since only duplex but not truly singlestranded DNA enhances the fluorescence of ethidium bromide, fluorescence after heating is a measure of covalently closed circular DNA remaining after reaction with endonuclease. Details concerning the ethidium bromide solution, heat denaturation, and fluorometry as well as the equations used to calculate the remaining fraction of supercoiled and relaxed DNAs were previously described (18).
Gel Electrophoresis-Electrophoresis of DNA through cylindrical gels (0.5 X 15 cm) of 0.7% agarose was performed in 36 nm Tris, 30 mM NaH2P04, 1 mM Na2EDTA (pH 7.7). In some cases samples were alkali-treated prior to electrophoresis (18) as described in the legend to Fig. 6. Electrophoresis was initiated at 95 V for 5 min and continued at 20 V for 16-20 h. Electrophoresis through 1.4% agarose gels containing 40 m~ Tris, 5 m~ sodium acetate, 1 mM NaZEDTA (pH 7.9) was performed at 110 V for 3 h for analysis of supercoiled DNA and for 2 h in gels containing 0.05 pg/ml of ethidium bromide for analysis of relaxed DNA. Staining of DNA in gels and photography of ethidium bromide fluorescence were previously described (18).
Mapping the Pst I Cleavage Site on PM2 DNA-Electrophoretic analysis (0.7% and 1.4% agarose gels) showed that digestion of Hpa 11-linearized PM2 DNA with Pst I produced two fragments corresponding to 0.87 and 0.13 map units in length. To determine on which side of the Hpa I1 site the enzyme cleaves (i.e. clockwise at 0.13 or counterclockwise at 0.87), an Hpa II/HindIII digest of PM2 DNA was treated with Pst I. The positions of the Hpa II/HindIII cleavages are known (20,21). The Hpa II/HindIII DNA fragment at M, =

RESULTS
Enzymatic Cleavage Rate of Supercoiled and Relaxed DNA as a Function of pH-A fluorometric assay for covalently closed circular DNA (18) was used to measure the rate of the first endonucleolytic cleavage (single or double strand break). Although the activity is optimal at acidic pH (7), cleavage of supercoiled DNA is readily measurable at neutral and alkaline pH values at elevated enzyme concentrations. Initial rate measurements with supercoiled DNA at pH 5 and pH 8 are shown in Fig. 1. The enzyme concentration at pH 8.0 (filled circles) is 75,000 times that at pH 5.0 (open circles). Providing less than 50% of the substrate was cleaved and chemical compounds which stabilize the activity at acidic pH values were present (see under "Materials and Methods"), the cleavage of closed circular DNA substrate was linear with time and enzyme concentration at all pH values tested.
The initial reaction velocity per enzyme molecule as a function of pH for supercoiled DNA and the same DNA converted to the relaxed form by topoisomerase I is shown in Fig. 2. The logarithm of activity declines as a relatively smooth and continuous function with increasing pH as expected for a single enzyme activity with acidic pH optimum. Supercoiled DNA is hydrolyzed at least 10,000-fold more rapidly than the   relaxed form (Table I). The maintenance of the highly preferential hydrolysis of supercoiled DNA suggests that the activity near neutral pH is due to the single strand specific nuclease itself and not a contaminating enzyme with neutral or alkaline pH optima. Even at pH 6.6, mung bean nuclease acts catalytically on supercoiled DNA, since 1 enzyme molecule cleaves 100 DNA molecules in 10 min (Fig. 2).

Hydrolysis Products of Supercoiled and Relaxed DNAs at Various pH
Values-Following incubation of DNA with enzyme a t 37 "C and pH values from 5 to 9, the hydrolysis products were analyzed by agarose gel electrophoresis. As shown in Fig. 3, with a supercoiled Fig. 5, nicked-circular DNA produced by mung bean nuclease at levels almost sufficient for complete hydrolysis of substrate (gel I ) and in 40-fold excess (gel 3) is converted to unit length linear molecules by phosphodiesterase (gels 2 and 4, respectively). No fragments of linear DNA are seen indicating that, even in the presence of excess mung bean nuclease, the nicked-circular DNA contains one nick per molecule. This conclusion was confirmed by alkali denaturation of the nickedcircular DNA prior to electrophoresis. The resulting singlestranded DNA separates into four bands of similar intensities as shown in Fig. 6, gel 2 After 10 min, reactions were stopped by cooling on ice. Samples 2 and 4 were treated with venom phosphodiesterase and the enzyme inactivated as described under "Materials and Methods." Ninety nanograms of DNA from each reaction mixture were applied to each gel and electrophoresis performed as described under "Materials and Methods. "  FIG. 6 (right). Electrophoresis of alkali-treated PM2 DNA on 0.7% agarose gels. I, supercoiled DNA, I', relaxed, closed circular DNA; SS, single-stranded linear and circular DNA. DNA (0.5 pg in 10 pl) was incubated at 37 "C with 0 (gel I ) , 0.05 (gels 2 and 3), or 2.0 (gels 4 and 5) units/rnl of mung bean nuclease in 10 m~ Tris-HCI (pH 7.0). After 10 min, reactions were terminated by cooling to 21 "C and adding Na2EDTA to 10 m~. Seven microliters of a solution containing 71 mM Tris-HC1 (pH 8.0), 26 m~ magnesium acetate, 71 mM nicotinamide adenine dinucleotide, and 0.14 mg/rnl of bovine serum albumin were added to each sample. Two microliters of E. coli DNA ligase (0.4 unit) were added to samples 3 and 5 while 2 p1 of ligase diluent (10 mM Tris, pH 8.0, 5 mM magnesium acetate, 25 p~ nicotinamide adenine dinucleotide, 50 pg/ml of serum albumin) was added to the remaining samples. After 20 min at 21 "C, Na2EDTA was added to 10 m~, and NaOH was added to 0.1 M. After 5 min at 21 "C, samples (containing 125 ng of DNA) were cooled to -0 "C, made 5% in glycerol and 0.01% bromphenol blue, and loaded under cold  3.53, 1.42, 0.61, 0.265, 0.245, 0.18, 0.06 (21). 6.30 (21); ECO RI- ADNA, 4.79,3.73,3.59, 3.07,2.18 (36 at 0.33, 0.54, 0.59,0.61, 0.63,0.72, and 0.76 map units (16). The open circles indicate the sites of early denaturation at 0, 0.16,  0.24, 0.28, 0.32, 0.64, 0.75, and 0.79 map units (16).

DISCUSSION
Though the optimal pH is acidic, catalytic quantities of mung bean nuclease efficiently cleave supercoiled PM2 DNA under neutral pH conditions. The reaction is highly preferential for supercoiled DNA, since 28,000 times more enzyme is required to cleave the relaxed toposiomer at the same rate. Supercoiled DNA is converted to singly nicked circular DNA. The nicked-circular DNA is resistant to linearization in the presence of at least 40 times the amount of enzyme required for complete conversion. The efficiency and limited nature of this reaction makes mung bean nuclease a useful alternative to pancreatic DNase I for the preparation of singly nicked PM2 DNA molecules. Since the nicks possess 3'-OH and 5"P termini, these molecules serve as substrates for DNA ligase (see under "Results"), exonuclease 111, and DNA polymerase. 2 It is interesting that the single strand specific endonuclease activity of venom phosphodiesterase shows a similar preference (10,000-fold) for supercoiled over relaxed PM2 DNA (22). In contrast to mung bean nuclease hydrolysis of supercoiled DNA, however, the nicked-circular DNA produced does not accumulate but is rapidly linearized, presumably facilitated by gap formation by the associated 3' "+ 5' exonuclease activity. Since efficient linearization occurs whether the initial nick is generated by the enzyme itself, by DNase I (22), or by mung bean nuclease, venom phosphodiesterase appears to be an excellent reagent for specific cleavage of the strand opposite nicks containing 3'-OH and 5'-P termini in duplex DNA.
The mung bean nuclease nicks in supercoiled PM2 DNA occur at three specific sites at a frequency at one nick per DNA molecule with no apparent strand specificity. Since several previous studies with supercoiled DNA showed a correlation between sites reactive to single strand specific agents and sites of early denaturation (16,(25)(26)(27), it was interesting to compare the locations of the mung bean nuclease cleavages to the locations of the early denaturation sites determined by Brack et al. (16). As illustrated in Fig. 8, mung bean nuclease prefers two sites (filled arrows) which map closely to each other (0.75 and 0.76 map units) and to one of the eight early denaturation sites (open circles). No cleavages are detected at the remaining seven early denaturation sites. A minor site for nicking occurs at 0.82 (Fig. 8, open arrowhead) which does not correspond to any known early denaturation site. Under different reaction conditions than those used here, the single strand specific endonuclease activity of venom phosphodiesterase (22) cleaves PM2 DNA at three sites (0.15, 0.62, and 0.78) which correlate with early denaturation sites and at two sites (0.72 and 0.85) which do not (27). Preliminary results with mung bean nuclease indicate that the positional specificity of cleavage is extremely sensitive to ionic condition^.^ Thus, the lack of correspondence of the sites cleaved by the two enzymes may reflect the differences in reaction conditions as well as possible differences in enzyme specificity. In any case, these studies indicate that the property of early denaturation alone is not a sufficient criterion to predict the sites recognized by these single strand specific endonucleases in supercoiled PM2 DNA. In contrast to these results obtained using enzymatic probes, single strand DNA binding protein (bacteriophage T4 gene 32 product) sites map exclusively at each of the eight early melting regions of PM2 DNA (16). Since mung bean nuclease preferentially cleaves DNA at dA 4 pN and dT J pN (l), and since early denaturation sites are presumed to be rich in dA + dT (28, 29), the base preference of the enzyme can not explain why most early denaturation sites are not cleaved. The discrimination between early melting sites by single strand specific endonucleases and the cleavage of additional sites not recognized by single strand DNA binding protein suggest that properties other than or in addition to early denaturation are involved in the recognition of supercoiled DNA by single strand specific endonucleases. Thus, sites other than dA + dT-rich regions may be the preferred locations of strand unwinding and separation in supercoiled PM2 DNA under our conditions.
Other sites of single strand character in negatively supercoiled DNA are possible. Recently, single strand specific endonucleases SI (30-32) and the T 7 gene 3 product (32) have been found to cleave some negatively supercoiled DNAs in the nonbase-paired loops of potential cruciforms. Thus, inverted repeat sequences in DNA are possible recognition sites for mung bean nuclease. Another possible site is in unwound regions which might occur at junctions between right-and left-handed DNA helices. One type of left-handed DNA called Z-DNA can form in sequences of alternating purines and pyrimidines (33)(34)(35). Finally, kinks or bends at regions in highly supercoiled molecules where the DNA helix doubles back on itself may occur in specific regions and have been suggested as possible cleavage sites (15). DNA sequencing studies in progress will help us distinguish between some of these possibilities.